The Development of Mitochondrial Gene Editing Tools and Their Possible Roles in Crop Improvement for Future Agriculture

Abstract We are living in the era of genome editing. Nowadays, targeted editing of the plant nuclear DNA is prevalent in basic biological research and crop improvement since its first establishment a decade ago. However, achieving the same accomplishment for the plant mitochondrial genome has long been deemed impossible. Recently, the pioneer studies on editing plant mitogenome have been done using the mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. It is well documented that mitochondria play essential roles in plant development and stress tolerance, particularly, in cytoplasmic male sterility widely used in production of hybrids. The success of mitochondrial genome editing enables studying the fundamentals of mitochondrial genome. Furthermore, mitochondrial RNA editing (mostly by nuclear‐encoded pentatricopeptide repeat (PPR) proteins) in a sequence‐specific manner can simultaneously change the production of translatable mitochondrial mRNA. Moreover, direct editing of the nuclear‐encoding mitochondria‐targeted factors required for plant mitochondrial genome dynamics and recombination may facilitate genetic manipulation of plant mitochondria. Here, the present state of knowledge on editing the plant mitochondrial genome is reviewed.

1.1 I suggest to change the title to something catchier, like "The development of mitochondrial gene editing tools and their possible roles in crop improvement for future agriculture".
In that aspect and based on their title, the authors need to emphasis in the first paragraph of the Intro part the importance of genome editing to plant improvement in light of climate changes, herbicidal and pesticidal uses (as e.g. BT toxin, glyphosates), as so far, the vast majority of crops have been made by "non-GMO" breeding strategies. What is the added value and importance of mtDNA add to that of the on-going efforts of nuclear gene transfer? (maybe one possibility is higher expression or the fact that these organelles are very often transmitted only maternally?!) I found several language mistakes and syntax errors that need to be fixed before publication. Some examples are found here: 1m1. Intro first paragraph: as outlined above start with the importance of genome editing to plant improvement 1m2. Page 4 line 17: "Scientists often say we are living in the era of genome editing." Consider to rephrase 1m3. Page 4 Line 20: replace feat with fate?
1m4. Page 4 Line 21: Mitochondrial DNA = mitogenome, or mtDNA 1m5. Page 4 Line 27: remove the in the quotes 1m6. Page 5 Lines 46-48: nuclear genome (3). In general, mitochondrial genomes are double-stranded DNA molecules differing in size and architecture (circular, linear or branched forms in different species) (4) -consider to rephrase 1m7. Page 5 Line 49: instead of "differ" in size write range in size.
1m8. Page 5 Line 57: instead of "varied genome size", consider "variations in mtDNA sizes" 1m9. Page 5 Line 58: instead of "known genes found in different terrestrial" consider " found in the mitogenomes of different…" 1m10. Page 6 Lines 63-64: "mitochondrial genome, the enormous efforts" consider "intensive" instead.. 1m11. Page 6 Line 104: replace it with "this system" 1m12. Page 10 line 160: "how to find a reliable method to deliver gRNA to the mitochondrial.." consider "to introduce an efficient mean to deliver the gRNA.." 1m13. Also the authors need to refer to the MSs: Sultan et al (2016) Plant cell, 28, 2805-2829and Val et al (2011 were the authors used tRNA-like ribozymes to be imported into plant mitochondria! 1m14. Page 12 line 192: mutant names (i.e. msh1) should be in Italic

Reviewer #2
Review for Advanced Genetics paper (Review GGN-2021-0011): The paper 'Editing the plant mitochondrial genome enables variations for crop improvement' by Yang et al. reviews most attempts of editing the plant mitochondrial genome, from mitochondrial DNA editing to targeted mitochondrial RNA editing, and that of nuclear factors involved in the mitochondrial genome dynamics and recombination. 2.2 My biggest criticism is that, although this review is concise, it is not very helpful as the text lacks clarity and structure. This may be due to an imprecise command of the English language, but the whole text is rather confusing.
• Has the author represented the main concepts and advances in the field fairly?
I think the description of the concepts and advances lacks precision and the information can be confusing in places.
Section 1: the introduction is generally fine.
Section 2 -the section about mtDNA editing is a bit confusing because the examples are alternatively from mammalian or plant cells, and there are substantial differences between them. I would prefer seeing a table summarising/comparing the different approaches tried in mammalian cells and in plants, and stressing the problems encountered.
Section 3 is very confusing because the authors mix different phenomena under the same name: in this section about "RNA editing", the authors describe the natural C -> U or U -> C RNA editing process performed by nuclear factors at a posttranscriptional level on mitochondrial and plastid RNAs by editing factors (PLS-type with DYW domains) and then cite the example of the "targeted modification of mitochondrial transcripts", which they also name RNA editing, using the P-type PPR protein RPF2 (which is not an editing factor). They then talk about the RNA editing assay performed by Physcomitrella editing factors by Oldenkott and collaborators in E. coli. This section should be rewritten, stressing what the purpose of the experiments was in each case (targeted modification of mitochondrial transcript expression, or straight editing assay etc…) and discuss their possible applications.
Section 4: The authors do not explain how manipulating nuclear factors involved in the mitochondrial genome dynamics and recombination could help precise editing the plant mitochondrial genome for crop improvement.
2.3 Section 5 (numbered 4 in the MS) is called perspectives but is more a summary of previous sections and has very few realistic perspectives as all these techniques are not really applicable to crop improvement at this stage. It should be improved.
• How will this Perspective lead researchers to conduct their research differently? I don't think this review is very helpful as it is.
• Are the claims, evidence and views presented in a clear and logical order?
Although the sections are presented in a logical order in the manuscript (DNA editing, "targeted mitochondrial transcript editing", nuclear genes), I think this review is very confusing because the contents of each sections are not sufficiently structured.
Minor points: 2m1-Line 79-85, please specify that this was done in murine cell cultures: "Hussain et al were able to perform gene editing of the mouse mitochondrial DNA in cell cultures" 2m2-Line 93: "F1F0 ATP synthase" should be "F1Fo ATP synthase") 2m3-Line 99: Nuclear mitochondrial DNA (NUMT) result from duplication of some regions of the mitochondrial DNA which are integrated to the nuclear genome, but are not, at least in the case of Arabidopsis thaliana, expressed. It is therefore very imprecise to describe this phenomenon as "During co-evolution of nuclear and mitochondrial genomes, the nuclear genome sometimes may develop pseudo-genes that have a high similarity to true mitochondrial genes". I think this should be rephrased. Nevertheless, this comment (line 96-102) is not very relevant in the context of this review.

Clarified current examples
Added the Mackenzie group research on using msh1 for crop improvement Added more discussion 1.1 I suggest to change the title to something catchier, like "The development of mitochondrial gene editing tools and their possible roles in crop improvement for future agriculture". In that aspect and based on their title, the authors need to emphasis in the first paragraph of the Introduction part the importance of genome editing to plant improvement in light of climate changes, herbicidal and pesticidal uses (as e.g. BT toxin, glyphosates), as so far, the vast majority of crops have been made by "non-GMO" breeding strategies. What is the added value and importance of mtDNA add to that of the on-going efforts of nuclear gene transfer? (maybe one possibility is higher expression or the fact that these organelles are very often transmitted only maternally?!) ED3 This is an excellent suggested title. The reviewer is right that the Introduction should set up what is possible with nuclear gene editing of crops and explain the need for mitochondrial editing (is plastid editing necessary or feasible?). This Introduction should set up the questions that the Perspectives section (ED2 above) answers and explains how crops can be improved using these new targeting techniques.

Agree
Title changed as suggested Added content to the introduction as suggested

Reviewer #1
The manuscript entitled "Editing the plant mitochondrial genome enables variations for crop improvement" by Jinghua Yang and his coauthors is review on the study of (plant) mitochondria genome editing. As note in the title, the authors aim to summarize the progress made in mitochondria genome editing and its importance to future crop improvement. The summary is suitable for publication and I have mostly minor comments for the authors.
1.1 I suggest to change the title to something catchier, like "The development of mitochondrial gene editing tools and their possible roles in crop improvement for future agriculture".
In that aspect and based on their title, the authors need to emphasis in the first paragraph of the Intro part the importance of genome editing to plant improvement in light of climate changes, herbicidal and pesticidal uses (as e.g. BT toxin, glyphosates), as so far, the vast majority of crops have been made by "non-GMO" breeding strategies. What is the added value and importance of mtDNA add to that of the on-going efforts of nuclear gene transfer? (maybe one possibility is higher expression or the fact that these organelles are very often transmitted only maternally?!) 1m8. Page 5 Line 57: instead of "varied genome size", consider "variations in mtDNA sizes" 1m9. Page 5 Line 58: instead of "known genes found in different terrestrial" consider " found in the mitogenomes of different…" 1m10. Page 6 Lines 63-64: "mitochondrial genome, the enormous efforts" consider "intensive" instead..
1m11. Page 6 Line 104: replace it with "this system" 1m12. Page 10 line 160: "how to find a reliable method to deliver gRNA to the mitochondrial.." consider "to introduce an efficient mean to deliver the gRNA.. Author Response: Revised them as suggested and marked them in red in the text.

Reviewer #2
The paper 'Editing the plant mitochondrial genome enables variations for crop improvement' by Yang et al. reviews most attempts of editing the plant mitochondrial genome, from mitochondrial DNA editing to targeted mitochondrial RNA editing, and that of nuclear factors involved in the mitochondrial genome dynamics and recombination.